Research reported in this publication was supported by the George F. Haddix President&#8217;s Faculty Research Fund and the National Institute of General Medical Science of the National Institutes of Health (NIH) under award number GM103427. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

<ol><li>Beuzón, C. R., Holden, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+mixed+infections+with+Salmonella+strains+to+study+virulence+genes+and+their+interactions+in+vivo%5BTitle%5D)+AND+%22Microbes+and+Infection%22%5BJournal%5D)">Use of mixed infections with Salmonella strains to study virulence genes and their interactions in vivo.</a> Microbes and Infection. 3 (14-15), 1345-1352 (2001).</li>
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<li>Schellenberg, J., Smoragiewicz, W., Karska-Wysocki, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+method+combining+immunofluorescence+and+flow+cytometry+for+improved+understanding+of+competitive+interactions+between+lactic+acid+bacteria+%28LAB%29+and+methicillin-resistant+S.+aureus+%28MRSA%29+in+mixed+culture%5BTitle%5D)+AND+%22Journal+of+Microbiological+Methods%22%5BJournal%5D)">A rapid method combining immunofluorescence and flow cytometry for improved understanding of competitive interactions between lactic acid bacteria (LAB) and methicillin-resistant S. aureus (MRSA) in mixed culture.</a> Journal of Microbiological Methods. 65 (1), 1-9 (2006).</li>
<li>Becker, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robust+Salmonella+metabolism+limits+possibilities+for+new+antimicrobials%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Robust Salmonella metabolism limits possibilities for new antimicrobials.</a> Nature. 440 (7082), 303(2006).</li>
<li>Rang, C., Galen, J. E., Kaper, J. B., Chao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fitness+cost+of+the+green+fluorescent+protein+in+gastrointestinal+bacteria%5BTitle%5D)+AND+%22Canadian+Journal+of+Microbiology%22%5BJournal%5D)">Fitness cost of the green fluorescent protein in gastrointestinal bacteria.</a> Canadian Journal of Microbiology. 49 (9), 531-537 (2003).</li>
<li>Santiviago, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+pools+of+targeted+Salmonella+deletion+mutants+identifies+novel+genes+affecting+fitness+during+competitive+infection+in+mice%5BTitle%5D)+AND+%22PLoS+Pathogens%22%5BJournal%5D)">Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.</a> PLoS Pathogens. 5 (7), e1000477(2009).</li>
<li>Ahmer, B. M., Gunn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+Salmonella+spp.+with+the+intestinal+microbiota%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Interaction of Salmonella spp. with the intestinal microbiota.</a> Frontiers in Microbiology. 2, 101(2011).</li>
<li>Gallagher, L. A., Shendure, J., Manoil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-scale+identification+of+resistance+functions+in+Pseudomonas+aeruginosa+using+Tn-seq%5BTitle%5D)+AND+%22MBio%22%5BJournal%5D)">Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq.</a> MBio. 2 (1), (2011).</li>
<li>Van Opijnen, T., Bodi, K. L., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tn-seq%3A+high-throughput+parallel+sequencing+for+fitness+and+genetic+interaction+studies+in+microorganisms%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms.</a> Nature Methods. 6 (10), 767(2009).</li>
<li>van Opijnen, T., Lazinski, D. W., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome%E2%80%90wide+fitness+and+genetic+interactions+determined+by+Tn%E2%80%90seq%2C+a+high%E2%80%90throughput+massively+parallel+sequencing+method+for+microorganisms%5BTitle%5D)+AND+%22Current+Protocols+in+Microbiology%22%5BJournal%5D)">Genome‐wide fitness and genetic interactions determined by Tn‐seq, a high‐throughput massively parallel sequencing method for microorganisms.</a> Current Protocols in Microbiology. 36 (1), (2017).</li>
<li>Mutalik, V. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual-barcoded+shotgun+expression+library+sequencing+for+high-throughput+characterization+of+functional+traits+in+bacteria%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria.</a> Nature Communications. 10 (1), 308(2019).</li>
<li>Yoon, H., Gros, P., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+PCR-based+competitive+index+for+high-throughput+screening+of+Salmonella+virulence+factors%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">Quantitative PCR-based competitive index for high-throughput screening of Salmonella virulence factors.</a> Infection and Immunity. 79 (1), 360-368 (2011).</li>
<li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proceedings of the National Academy of Sciences. 97 (12), 6640-6645 (2000).</li>
<li>Henard, C. A., Bourret, T. J., Song, M., Vázquez-Torres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+redox+balance+by+the+stringent+response+regulatory+protein+promotes+antioxidant+defenses+of+Salmonella%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Control of redox balance by the stringent response regulatory protein promotes antioxidant defenses of Salmonella.</a> Journal of Biological Chemistry. 285 (47), 36785-36793 (2010).</li>
<li>Poteete, A. R., Fenton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%BB+red-dependent+growth+and+recombination+of+phage+P22%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">λ red-dependent growth and recombination of phage P22.</a> Virology. 134 (1), 161-167 (1984).</li>
<li>McCollister, B. D., Bourret, T. J., Gill, R., Jones-Carson, J., Vázquez-Torres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repression+of+SPI2+transcription+by+nitric+oxide-producing%2C+IFN%CE%B3-activated+macrophages+promotes+maturation+of+Salmonella+phagosomes%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">Repression of SPI2 transcription by nitric oxide-producing, IFNγ-activated macrophages promotes maturation of Salmonella phagosomes.</a> Journal of Experimental Medicine. 202 (5), 625-635 (2005).</li>
<li>Shaw, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Salmonella+enterica+serovar+Typhimurium+has+three+transketolase+enzymes+contributing+to+the+pentose+phosphate+pathway%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Salmonella enterica serovar Typhimurium has three transketolase enzymes contributing to the pentose phosphate pathway.</a> Journal of Biological Chemistry. 293 (29), 11271-11282 (2018).</li>
<li>Freter, R., O'Brien, P., Macsai, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+chemotaxis+in+the+association+of+motile+bacteria+with+intestinal+mucosa%3A+in+vivo+studies%5BTitle%5D)+AND+%22Infection+and+immunity%22%5BJournal%5D)">Role of chemotaxis in the association of motile bacteria with intestinal mucosa: in vivo studies.</a> Infection and immunity. 34 (1), 234-240 (1981).</li>
<li>Taylor, R. K., Miller, V. L., Furlong, D. B., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+phoA+gene+fusions+to+identify+a+pilus+colonization+factor+coordinately+regulated+with+cholera+toxin%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin.</a> Proceedings of the National Academy of Sciences. 84 (9), 2833-2837 (1987).</li>
<li>Nogva, H. K., Dromtorp, S., Nissen, H., Rudi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ethidium+monoazide+for+DNA-based+differentiation+of+viable+and+dead+bacteria+by+5%27-nuclease+PCR%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR.</a> Biotechniques. 34 (4), 804-813 (2003).</li>
<li>Nocker, A., Camper, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+approaches+toward+preferential+detection+of+viable+cells+using+nucleic+acid+amplification+techniques%5BTitle%5D)+AND+%22FEMS+Microbiology+Letters%22%5BJournal%5D)">Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques.</a> FEMS Microbiology Letters. 291 (2), 137-142 (2009).</li>
<li>Nocker, A., Cheung, C. -Y., Camper, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+propidium+monoazide+with+ethidium+monoazide+for+differentiation+of+live+vs.+dead+bacteria+by+selective+removal+of+DNA+from+dead+cells%5BTitle%5D)+AND+%22Journal+of+Microbiological+Methods%22%5BJournal%5D)">Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells.</a> Journal of Microbiological Methods. 67 (2), 310-320 (2006).</li>
<li>Nocker, A., Mazza, A., Masson, L., Camper, A. K., Brousseau, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+detection+of+live+bacteria+combining+propidium+monoazide+sample+treatment+with+microarray+technology%5BTitle%5D)+AND+%22Journal+of+Microbiological+Methods%22%5BJournal%5D)">Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology.</a> Journal of Microbiological Methods. 76 (3), 253-261 (2009).</li>
<li>Nocker, A., Sossa, K. E., Camper, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+monitoring+of+disinfection+efficacy+using+propidium+monoazide+in+combination+with+quantitative+PCR%5BTitle%5D)+AND+%22Journal+of+Microbiological+Methods%22%5BJournal%5D)">Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR.</a> Journal of Microbiological Methods. 70 (2), 252-260 (2007).</li>
<li>Nocker, A., Sossa-Fernandez, P., Burr, M. D., Camper, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+propidium+monoazide+for+live%2Fdead+distinction+in+microbial+ecology%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">Use of propidium monoazide for live/dead distinction in microbial ecology.</a> Applied and Environmental Microbiology. 73 (16), 5111-5117 (2007).</li>
<li>Price-Carter, M., Tingey, J., Bobik, T. A., Roth, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Alternative+Electron+Acceptor+Tetrathionate+Supports+B12-Dependent+Anaerobic+Growth+ofSalmonella+enterica+Serovar+Typhimurium+on+Ethanolamine+or+1%2C+2-Propanediol%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">The Alternative Electron Acceptor Tetrathionate Supports B12-Dependent Anaerobic Growth ofSalmonella enterica Serovar Typhimurium on Ethanolamine or 1, 2-Propanediol.</a> Journal of Bacteriology. 183 (8), 2463-2475 (2001).</li>
<li>Taylor, R. G., Walker, D. C., McInnes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((E.+coli+host+strains+significantly+affect+the+quality+of+small+scale+plasmid+DNA+preparations+used+for+sequencing%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing.</a> Nucleic Acids Research. 21 (7), 1677(1993).</li>
<li>Cherepanov, P. P., Wackernagel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+disruption+in+Escherichia+coli%3A+Tc+R+and+Km+R+cassettes+with+the+option+of+Flp-catalyzed+excision+of+the+antibiotic-resistance+determinant%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">Gene disruption in Escherichia coli: Tc R and Km R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant.</a> Gene. 158 (1), 9-14 (1995).</li>
</ol>

The authors have nothing to disclose.

The ability to accurately quantify microorganisms is of paramount importance to microbiology research, and the ability to enumerate unique strains from an initial mixed population has proved to be an invaluable tool for assessing fitness and virulence traits in bacteria. However, the techniques for accomplishing this have not progressed in pace with modern developments in molecular biology. The technology to easily modify the chromosomes of many bacteria, including S. Typhimurium, has been available for nearly t...

Assessing fitness and virulence of pathogenic organisms is a fundamental aspect of microbiology research. It enables comparisons to be made between strains or between mutated organisms, which allows researchers to determine the importance of certain genes under specific conditions. Traditionally, virulence assessment utilizes an animal model of infection using different bacterial strains and observing the outcome of the infected animal (e.g. Infectious Dose50, Lethal Dose50, time to death, symptom severity, lack of symptoms, etc.). This procedure provides valuable descriptions of virulence, but it requires strains to cause considerable differences in outcomes in order to detect variations from wild-type. Furthermore, results are semi-quantitative because while disease progression and symptom severity can be subjectively quantified over time, interpretation of virulence compared to wild-type is more qualitative (i.e. more, less, or equally virulent). A common alternative to performing animal infectivity assays is to generate competitive indices (CIs), values that directly compare fitness or virulence of a strain to a wild-type counterpart in a mixed infection1. This technique has numerous advantages over a traditional animal model of infection by standardizing virulence to a wild-type strain and determining a quantifiable value to reflect the degree of attenuation. This technique can also be adapted to analyze gene interactions in bacteria by determining a canceled out competitive index (COI)2. Calculating a COI for a group of mutated organisms allows researchers to determine whether two genes independently contribute to pathogenesis or if they are involved in the same virulence pathway and dependent on each other. Additionally, calculating a CI requires enumeration of bacteria which can provide valuable insights into the pathogenesis of organisms. CIs and COIs also allow researchers to asses avirulent strains that do not cause clinical disease but still have differences in fitness. This technique is limited by the use of traditional antibiotic resistance markers to identify strains, thereby limiting the number of input strains to only one or two at a time. Because of this limitation, large numbers of experimental groups and replicates are required, which in addition to adding to labor and material costs, also increases opportunities for variability in experimental conditions and inaccurate results. (For a thorough review of the benefits and applications of using mixed infections to study virulence, fitness, and gene interactions, see C.R. Beuz&#243;n and D.W. Holden 1)
Attempts have been made to overcome this limitation, such as the use of fluorescently-labeled cells quantified via flow cytometry3,4,5. This technique quantifies cells using either 1) labeled antibodies to phenotypic markers or 2) endogenously produced fluorescent proteins. The use of labeled antibodies has a limit of detection of 1,000 cells/mL, and therefore requires a high number of cells to analyze3. Cells expressing fluorescent proteins have an altered physiology and are susceptible to fitness changes resulting from high protein expression6. Both methods are limited by the number of fluorescent markers detectable using flow cytometry. An advancement in molecular quantification was achieved through the development of a microarray technique that detected attenuation in 120 strains from an initial mixed infection of over 1,000 strains in a murine model7. This technique utilized a microarray analysis of RNA from mutated strains, which lead to considerable variability in the outcome.&#160; Nevertheless, it established that large pools of mixed infections can be a useful tool and that by utilizing sensitive detection techniques, differences in bacterial virulence can be identified. With the development of the next generation sequencing, Tn-seq expanded the utility of transposon mutations, enabling a powerful method for quantifying bacteria that were randomly mutated8,9,10,11. An alternative protocol was recently developed that eliminates the need for transposons and instead uses DNA barcodes to more easily identify and track genomic changes and their impact on fitness12. This technology is a major advancement, but the insertion of the genomic barcodes is still a random process. To overcome the randomness of previous experiments, Yoon et al. developed a method to calculate the CIs of Salmonella strains using unique DNA barcodes inserted at precise locations on the chromosomes of bacteria13. Unique barcoded strains were detected using a qPCR-based method with SYBR green and primers specific to each unique barcode. The technique was limited by constraints imposed by qPCR, including differences in primer efficiencies and low sensitivity, evidenced by the need for nested-PCR prior to qPCR. Nevertheless, this approach demonstrated that targeted genomic modifications could be exploited for detecting and potentially quantifying pools of multiple bacterial strains.
In the following protocol, we describe a novel methodology to perform bacterial competition experiments with large pools of mixed inocula followed by accurate quantification using a highly sensitive digital PCR technique. The protocol involves genetically-labeling bacterial strains with a unique DNA barcode inserted on an innocuous region of the chromosome. This modification allows strains to be quickly and accurately quantified using modern molecular technology instead of traditional serial dilutions, replica plating, and counting colony forming units that rely on phenotypic markers (i.e. antibiotic resistance). The modifications allow for simultaneous assessment of many strains in a single pooled inoculum, substantially reducing the possibility of experimental variability because all strains are exposed to the exact same conditions. Furthermore, while this technique was developed in Salmonella enterica serovar Typhimurium, it is highly adaptable to any genetically malleable organism and nearly any experimental design where accurate bacterial counts are required, providing a new tool to increase accuracy and throughput in microbiology laboratories without the constraints imposed by previous methods.

<table><tbody><tr><td>1.5 mL microcentrifuge tubes</td><td>Eppendorf</td><td>22600028</td><td>Procure from any manufacturer</td></tr><tr><td>16 mL culture tubes</td><td>MidSci</td><td>8599</td><td>Procure from any manufacturer</td></tr><tr><td>5-200 &amp;mu;L pipette tips</td><td>RAININ</td><td>30389241</td><td>Procure alternative tip brands with caution based on manufacturing quality</td></tr><tr><td>5-50 &amp;mu;L multichannel pipette</td><td>RAININ</td><td>17013804</td><td>Use alternative multichannel pipettes with caution</td></tr><tr><td>Agarose</td><td>ThermoFisher Scientific</td><td>BP160-500</td><td>Procure from any manufacturer</td></tr><tr><td>BLAST Analysis</td><td>NCBI</td><td>N/A</td><td>https://blast.ncbi.nlm.nih.gov/Blast.cgi</td></tr><tr><td>C1000 Touch Thermocycler with 96-Deep Well Reaction Module</td><td>Bio Rad</td><td>1851197</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>Chemically competent DH5&amp;alpha;</td><td>Invitrogen</td><td>18258012</td><td>Procure from any manufacturer or prepare yourself</td></tr><tr><td>Chloramphenicol</td><td>ThermoFisher Scientific</td><td>BP904-100</td><td>Procure from any manufacturer</td></tr><tr><td>Cytation5 Microplate reader</td><td>BioTek</td><td>CYT5MF</td><td>Procure from any manufacturer, use any system capable of accurately quantifying DNA</td></tr><tr><td>Data Analysis Software (QuantaSoft and QuantaSoft Data Analysis Pro)</td><td>Bio Rad</td><td>N/A</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>ddPCR 96-Well Plates</td><td>Bio Rad</td><td>12001925</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>ddPCR Droplet Reader Oil</td><td>Bio Rad</td><td>1863004</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>ddPCR Supermix for Probes (No dUTP)</td><td>Bio Rad</td><td>1863024</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>DG8 Cartridges for QX200/QX100 Droplet Generator</td><td>Bio Rad</td><td>1864008</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>DG8 Gaskets for QX200/QX100 Droplet Generator</td><td>Bio Rad</td><td>1863009</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>Droplet Generation Oil for Probes</td><td>Bio Rad</td><td>1863005</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>Kanamycin</td><td>ThermoFisher Scientific</td><td>BP906-5</td><td>Procure from any manufacturer</td></tr><tr><td>Luria-Bertani agar</td><td>ThermoFisher Scientific</td><td>BP1425-2</td><td>Procure from any manufacturer or make it yourself from agar, tryptone, yeast digest, and NaCl</td></tr><tr><td>Luria-Bertani broth</td><td>ThermoFisher Scientific</td><td>BP1426-2</td><td>Procure from any manufacturer or make it yourself from tryptone, yeast digest, and NaCl</td></tr><tr><td>PCR Plate Heat Seal, foil, pierceable</td><td>Bio Rad</td><td>1814040</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>PCR Tubes</td><td>Eppendorf</td><td>951010022</td><td>Procure from any manufacturer</td></tr><tr><td>Petri dishes</td><td>ThermoFisher Scientific</td><td>FB0875712</td><td>Procure from any manufacturer</td></tr><tr><td>pPCR Script Cam SK&lt;sup&gt;+&lt;/sup&gt;</td><td>Stratagene/Agilent</td><td>211192</td><td>No longer available commercially</td></tr><tr><td>Primer/Probe Design</td><td>IDT</td><td>N/A</td><td>https://www.idtdna.com/Primerquest/Home/Index</td></tr><tr><td>pSKAP and pSKAP_Barcodes</td><td>Addgene</td><td>Plasmid numbers 122702-122726</td><td>www.addgene.org</td></tr><tr><td>PX1 PCR Plate Sealer</td><td>Bio Rad</td><td>1814000</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>QX200 Droplet Generator</td><td>Bio Rad</td><td>1864002</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>QX200 Droplet Reader</td><td>Bio Rad</td><td>1864003</td><td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td></tr><tr><td>&lt;em&gt;Salmonella typhimurium&lt;/em&gt; strain ATCC 14028s</td><td>ATCC</td><td>ATCC 14028s</td><td>www.atcc.org</td></tr><tr><td>Take3 Micro-Volume Plate</td><td>BioTek</td><td>TAKE3</td><td>Procure from any manufacturer, use any system capable of accurately quantifying DNA</td></tr><tr><td>Thermo Scientific FastDigest BamHI</td><td>ThermoFisher Scientific</td><td>FERFD0054</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific FastDigest DpnI</td><td>ThermoFisher Scientific</td><td>FERFD1704</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific FastDigest HindIII</td><td>ThermoFisher Scientific</td><td>FERFD0504</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific GeneJet Gel Extraction and DNA Cleanup Micro Kit</td><td>ThermoFisher Scientific</td><td>FERK0832</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific GeneJet Miniprep Kit</td><td>ThermoFisher Scientific</td><td>FERK0503</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific Phusion High-Fidelity DNA Polymerase</td><td>ThermoFisher Scientific</td><td>F534L</td><td>Procure from any manufacturer</td></tr><tr><td>Thermo Scientific T4 DNA Ligase</td><td>ThermoFisher Scientific</td><td>FEREL0011</td><td>Procure from any manufacturer</td></tr><tr><td>Thermocycler</td><td>Bio Rad</td><td>1861096</td><td>Procure from any manufacturer</td></tr><tr><td>UVP Visi-Blue Transilluminator</td><td>ThermoFisher Scientific</td><td>UV95043301</td><td>Or other transiluminator that allows visualization of DNA</td></tr><tr><td>Water, Molecular Biology Grade</td><td>ThermoFisher Scientific</td><td>BP28191</td><td>Procure from any manufacturer</td></tr></tbody></table>

digital pcr-based competitive index for high-throughput analysis of fitness in salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

The use of this methodology requires that appropriate control reactions are performed to validate the sensitivity and specificity of each probe used to identify target DNA. In this representative experiment, we validated eight unique DNA barcodes with the eight corresponding probes for identification. All eight probes had a low rate of false positives in both NTC and negative control reactions (Table 3), highlighting their specificity even among highly similar DNA sequenc...

Watch this Scientific Journal Video about Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella at JoVE.com

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

digital-pcr-based-competitive-index-for-high-throughput-analysis

Research

JoVE Journal

Genetics

9.6K Views.  Creighton University School of Medicine. This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

Video: Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

Environment

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

Immunology and Infection

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription factors, histone modifications, and other DNA-associated proteins. ChIP-seq data can also be used to find differential binding of transcription factors in different environmental conditions or cell types. Initially, ChIP was performed through hybridization on a microarray (ChIP-chip); however, ChIP-seq has become the preferred method through technological advancements, decreasing financial barriers to sequencing, and massive amounts of high-quality data output. Techniques of performing ChIP-seq with bacterial biofilms, a major source of persistent and chronic infections, are described in this protocol. ChIP-seq is performed on Salmonella enterica serovar Typhimurium biofilm and planktonic cells, targeting the master biofilm regulator, CsgD, to determine differential binding in the two cell types. Here, we demonstrate the appropriate amount of biofilm to harvest, normalizing to a planktonic control sample, homogenizing biofilm for cross-linker access, and performing routine ChIP-seq steps to obtain high quality sequencing results.

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) is a method used to establish interactions between transcription factors and the genomic sequences they control. This protocol outlines techniques for performing ChIP-seq with bacterial biofilms, using Salmonella enterica serovar Typhimurium bacterial biofilm as an example.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Biology

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Modified Most Probable Number Assay to Quantify Salmonella in Raw and Ready-to-Cook Chicken Products

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella focus on prevalence rather than quantification, which limits their utility in assessing contamination levels and risks. This study introduces a novel most probable number (MPN) assay designed to quantify Salmonella in ready-to-cook poultry products, such as chicken cordon bleu. The method involves washing the poultry sample, concentrating the rinse through centrifugation, and serially diluting it in a 48-well block. The MPN assay is integrated with the loop-mediated isothermal amplification (LAMP) method to provide a sensitive, accurate, and rapid quantification of Salmonella contamination within the same timeframe as existing Food Safety and Inspection Service (FSIS) protocols. Results show a strong linear correlation between the MPN-LAMP measurements and theoretical inoculation levels (R&#178; = 0.933). However, variability at lower concentrations highlights challenges in accurately detecting Salmonella at these levels, with the practical lower limit of detection estimated at approximately 300 CFU/g. Potential refinements to improve the protocol's applicability include increasing the quantity sampled to further improve the limit of detection, optimizing enrichment media formulations, and expanding molecular detection to target multiple Salmonella serovars. Overall, this study presents a practical tool for the food industry, enabling reliable quantification of Salmonella contamination in poultry products, contributing to improved food safety and public health.

Accurately quantifying Salmonella in poultry at low levels is a current industrial and regulatory challenge. This protocol describes an MPN assay that enables quantification of Salmonella in raw and ready-to-cook poultry products. This method is fast, sensitive, and aligns with FSIS guidelines, enhancing food safety and supporting public health efforts.

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella ...

Duplex Digital PCR for Simultaneous Quantification of Dual Genetic Markers

Source: Cao, Y., et al. <a href="https://app.jove.com/v/53611/">A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the duplex digital PCR (ddPCR) technique &#8212; a modification of the traditional PCR technique that is useful in detecting two different genetic markers simultaneously. A single PCR reaction is partitioned into nanoliter-sized emulsified droplets that are independently amplified, and the detection of differently colored fluorescence amplification signals from the fraction of droplets is used to compute the initial concentration of the target sequences.

Source: Cao, Y., et al. A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Summary

Explore More Videos

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Competitive Genomic Screens of Barcoded Yeast Libraries

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Modified Most Probable Number Assay to Quantify Salmonella in Raw and Ready-to-Cook Chicken Products

Duplex Digital PCR for Simultaneous Quantification of Dual Genetic Markers